PROJECT SUMMARY Sepsis is a life-threatening systemic inflammatory host response to microbial infection that affects in excess of 700,000 patients annually in the US alone, with current in-hospital mortality of septic patients still reaching about 25%. Current treatments are limited to control of infection with antibiotics and intensive supportive care to sustain organ function, and new therapies are urgently needed. Many candidate drugs showed promise in preclinical animal studies, but failed to improve, or even worsened outcomes in clinical studies. This has raised general concerns about a profound lack of understanding of sepsis pathology, and the inadequacy of animal studies to mimic human disease and predict clinical efficacy. This project takes a radically different approach to address these concerns and is based on the seminal observation that a naturally occurring mutation in human blood coagulation factor V (fV Leiden) protects against death from infection in humans, as well as in various mouse models of sepsis and lethal sterile inflammation. Our preliminary studies indicate that the natural survival advantage of fV Leiden carriers is mediated by the ability of endogenous activated protein C (aPC) to trigger a molecular switch in the mode of signal transduction by protease-activated receptor 2 (PAR2). This switch modulates in an as yet unknown manner the immune-regulatory function of a distinct, infection-elicited population of innate immune cells to enable the resolution of inflammation and improve survival. The objective of the proposed studies is to delineate the cellular and molecular mechanisms that protect heterozygous carriers of this mutation from death by sepsis, and apply this knowledge to instruct therapeutic approaches targeting these natural survival pathways. This is accomplished in three specific aims: (1) To delineate the biological responses of infection- elicited myeloid innate immune cells that are controlled by two alternate modes of PAR2 signaling, and how these responses modify the overall outcome of infection. (2) To determine how the natural survival mechanisms operating in fV Leiden carriers can be engaged in normal mice by therapeutic administration of recombinant variants of aPC that selectively target these pathways without causing bleeding complications. (3) To determine whether the novel molecular and cellular immune mechanisms discovered in mice also are relevant to the human host response to infection. Our studies will thereby document previously unknown mechanisms by which coagulation receptor signaling shapes the innate immune response to infection. These novel pathways can explain the limited efficacy of recombinant aPC in past clinical trials, and also instruct a rationale approach for greatly improving the clinical efficacy of therapies with aPC or novel reagents with aPC-like activity.